The objective for the Principal Investigator of this Research Career Award is to evolve into an independent basic science investigator in renal pathophysiology. The applicant is a pediatric nephrologist working in the Departments of Medicine and Pediatrics at the Yale University School of Medicine. Both departments have a strong commitment to the development of young scientists. Through these departments the applicant will have a focused program of didactic courses, participation in research seminars, and protected research time. The research project detailed here has been designed to provide an excellent training vehicle for furthering the applicant's transition to an independent investigator. The specific aims outlined for this project provide the applicant with the opportunity to develop expertise in multiple laboratory techniques, the ability to refine experimental design and data interpretation skills, and continued supervision by her mentor, Dr. Peter Aronson. The proposed project concentrates on the role of phosphorylation in the regulation of NHE3, a principal apical membrane Na/H exchanger in the proximal tubule. This exchanger is responsible for the majority of sodium bicarbonate reabsorption by the kidney and therefore plays a vital role in acid-base and volume homeostasis. The applicant has successfully generated phosphospecific antibodies to two different putative phosphorylation sites on the C-terminus of NHE3, serines 552 and 605. She proposes to study NHE3 phosphorylation at each of these sites in response to various physiologic and pharmacologic maneuvers. These antibodies will then be used to determine the subcellular localization of phosphorylated NHE3 at baseline and in response to various stimuli known to alter NHE3 activity. Finally, associated NHE3 proteins interacting specifically with either phosphorylated or non-phosphorylated NHE3 will be sought, and any identified interactions further characterized with regard to NHE3 function and distribution. The experiments in this proposal will deepen our understanding of NHE3 phosphorylation as a regulatory mechanism, and thereby contribute to understanding disorders characterized by abnormal volume and acid-base balance, such as hypertension and osteoporosis.